The AllPrep RNA/Protein Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

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Highly intact RNA and high-quality protein from the same cell sample.|AllPrep RNA/Protein procedure.|Purification of functional protein.|Sensitivity in real-time RT-PCR.|

Total RNA was purified from HeLa cells and analyzed on the Agilent 2100 Bioanalyzer. The rRNA ratio (28S/18S) was 2.04. Total protein was purified from HeLa cells and analyzed by western blot. Analysis of duplicate protein samples with antibodies against Lamin (numclear protein) and TIM23 (mitochondrial protein) demonstrates that the total protein fraction contains proteins from subcellular compartments.||HeLa cells in a 96-well plate were transfected with a β-galactosidase-reporter plasmid. After 48 hours, protein was purified and β-galactosidase activity was assayed (yellow indicates enzyme activity). Protein was purified using either the AllPrep RNA/Protein Kit (AllPrep) or the RNeasy Mini Kit (cell lysate was passed through an RNeasy spin column, and protein was precipitated from the flow-through) (Other). Each column of wells shows triplicate results from one cell sample.|RNA was purified from HeLa cells using the AllPrep RNA/Protein Kit and reverse-transcribed. Serial dilutions of cDNA equivalent to 100 pg to 100 ng of total RNA were analyzed using a real-time RT-PCR assay for human GSK3B (glycogen synthase kinase 3 beta). Reactions were performed in duplicate. RNA purified using the RNeasy Mini Kit showed similar CT values and reproducibility (data not shown). NTC: No template control.|

The AllPrep RNA/Protein Kit is designed to purify total RNA and native protein simultaneously from the same sample of cultured cells. Analysis of purified RNA from a sample of cells on the Agilent 2100 Bioanalyzer typically gives RIN values of 10, indicating intact RNA. The native protein purified from the same cell sample contains all cellular proteins (see figure “Highly intact RNA and high-quality protein from the same cell sample”). RNA purified with the AllPrep RNA/Protein Kit is suitable for real-time RT-PCR (see figure "Sensitivity in real-time RT-PCR") and purified cellular proteins demonstrate functionality in downstream enzyme assays (see figure "Purification of functional protein").

Principle

The AllPrep RNA/Protein Kit is designed to purify total RNA and native protein simultaneously from the same sample of cultured cells. For experiments to validate siRNA-mediated knockdown at the mRNA and protein levels, the kit streamlines sample preparation and allows the number of cell cultures to be significantly reduced. In addition, knockdown analysis is more reliable since RNA and protein come from the same sample.The AllPrep RNA/Protein Kit is part of QIAGEN’s range of closely integrated products for RNAi and gene expression analysis. These products provide highly reproducible and sensitive results. Products include optimized PCR master mixes, ready-to-run assays, siRNAs, and transfection reagents. Standardization of RNAi and gene expression analysis workflows by these products have contributed to success in many areas of biomedical research.

Procedure

The spin-column–based procedure enables purification of RNA and protein from a single cell sample in less than 40 minutes (see flowchart "AllPrep RNA/Protein procedure"). A method is available for simultaneous purification of DNA, RNA, and protein (suitable for western blotting, not for enzyme assays) from cultured cells and tissues using the AllPrep DNA/RNA/Protein Kit.

Applications

The AllPrep RNA/Protein Kit allows purification of RNA and protein from a single cell culture. The purified RNA and protein provide high performance in downstream applications, such as real-time RT-PCR, western blotting, and enzyme assays. (Note: The functionality of any protein of interest in an assay has to be tested according to its properties.)